Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a

Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B. pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B. pseudomallei, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B. pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B. pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA.


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Aspects of CRISPR technology for diagnostics and treatment needs more advanced elaboration. https://link.springer.com/article/10.1007/s40097-022-00472-7 We appreciate the reviewer for the comment. We have re-written the paragraph concerning CRISPR-Cas (Line 100-127) so that it incorporates the reviewer's suggestion and appropriate citations.

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Define the "frequency" and the time duration for sonication in the method protocol. We apologize for being unclear. We have added sonication parameters in the Materials and Methods section.
• Report the length, melting temperature and probes of the primers used in the RPA and the region used for the amplification. We have provided the information of primers and probes in a supplementary table (Table S3) • Correct the "µl" spelling throughout the document We thank the reviewer for pointing this out. We have corrected the spelling throughout the document.

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Define the importance of the target sites "crBP34", "crBP36", "crBP38" and its role as a controlling factor for melioidosis. We thank the reviewer for helping us make this manuscript clearer. All eight candidates are located in both coding and non-coding regions of the genome. Unfortunately, functions of many of these loci in melioidosis are not well characterized. Nonetheless, we have edited Fig 1D to include genomic location, annotation and product description of encoded protein (if any) for all final candidates.

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Are the researchers making any modifications in the purification protocol, as there is a lack of component "sodium phosphate" in both the binding and eluting buffer. The researchers are requested to share the purification profile As suggested by the reviewer, we have made the purification protocol clearer by detailing the previously shortened steps and providing all components of buffers. Please note that, we have used Tris/NaCl-based buffers for all our purification protocols. • "The results showed that all three crRNAs could specifically detect B. pseudomallei, but not other pathogens". Are the authors taking any range level to specify the detection limit, as it is observed form the Figure 2B: the fluorescence intensity is detected for the "S. epidermidis" and "B. thailandensis" as well apart from "B. pseudomallei"; and in Figure 2C: the fluorescence intensity is detected for the "S. pneumoniae" and "S. aureus ATCC" also on a lower scale. The reviewer has raised a great point. This issue likely stemmed from the distorted unit on the logarithmic scale (log10), which resulted in the disproportionately elevated signals of S. epidermidis and B. thailandensis in the previous Figure 2B. We became aware that a logarithmic plot may have caused a confusion and thus replotted the graph on a linear scale (new Figure 2B-D). This will also enable readers to better differentiate the kinetics of each crRNA.
New Figure 2B-D Reviewer #2: The authors had robust methods that were thoroughly explained. My concerns/questions regarding the methods are listed below.
Line 136: Define LB. Was Lennox broth or Luria Bertani broth used? Line 290 defines LB as Luria-Bertani but it would be useful to have LB defined on line 136 as well. We have clarified that our LB was Luria Bertani, as suggested.
It was unclear to me how the authors selected crBP34, 36 and 38 from the list of candidate target sites from Fig 1D. We thank the reviewer for this helpful comment. To clarify this, we have elaborated more on this issue (Line 411-415). The criterion was simply based on how well these target sites were amplified during RPA. We also have provided these data in Fig S1.   Fig 1 was a very helpful figure to understand the design process for the Bp specific CRISPR RNA. I was happy to see that the authors screened the possible CRISPR sites against cross-reactive pathogens and I have two questions about this step. 1) Why did the authors not in-silico screen the final target (crBP34) against all available bacterial pathogens from NCBI. 2) If the ultimate goal of this assay is to test clinical samples why were the targets not screened against the human genome to look for interactions? We are very pleased to learn that the reviewer found Figure 1 useful and would like to thank them for their kind encouragement.
We agree with the reviewer that a more comprehensive screening would be to include all available bacterial pathogen and human genomes from NCBI in the initial scan. However, our filtering criteria was site-specific and computationally expensive. To ensure specificity, the sequence of TTTVN1N2N3…N20 in Bukholderia pseudomallie must not be detected in other species with either less than four mismatches in position N7-N20 or less than three mismatches in position N1-N6 or less than two consecutive mismatches in position N1-N6. This resulted in a highly stringent set of candidates at the expense of computation load and led us to limit an initial search to a smaller subset of common pathogens found in the same clinical setting as Burkholderia pseudomallei to ensure no cross-reaction.
In light of the reviewer's comment, we performed a post-hoc analysis to screen for the specificity of eight crRNAs targets against bacteria and human genomes. To reduce computational load, we used a WebBLAST before further screening the candidate (see item 5 in Materials and Methods) for their potential cross-reacting sites using a more relaxed threshold, namely relaxing 4 th position of PAM and allowing 0-1 mismatch in N1-N20 (deduced from studies in Ref 1 and 2). We found no match in any of the 40,827 archived bacterial reference genomes and in human reference genome GRCh38.p13 from NCBI. The analysis is summarized as Fig S3 and S4.
Besides, the high specificity of crRNAs, RPA primers also help add another layer of specificity to the DETECTR platform, as target sites must be able to be amplified before the CRISPR reaction.
Consistently, our data presented in Fig 2B-D showed that our detection platform with crBP34, 36 and 38 show no cross-reaction with genomic DNA from tested bacteria pathogens as well as from human (T84 cell line).